We have proposed a cross-bridge model of muscle contraction in which, during each of ATP hydrolysis, the myosin cross-bridge alternates between a conformation in which the cross-bridge binds strongly to actin, and a conformation in which the cross-bridge binds weakly to actin and is in rapid equilibrium between attached and detached cross-bridge states. It is while the cross-bridge is in the latter conformation that ATP hydrolysis and a separate rate-limiting occur. In the present study we have tested several predictions of this model both in vitro and in single skinned rabbit muscle fibers. First, we have measured 0-18 exchange using both myosin subfragment-1 (S-1) from skeletal muscle and from cardiac muscle. Earlier results with skeletal muscle acto-S-1 suggested that bound Pi may not have complete freedom of rotation of the active site of S-1 which, in turn may limit the rate of 0-18 exchange. Our results with cardiac S-1 strongly support this conclusion. Furthermore, they demonstrate that there must be a separate ATP hydrolysis step and rate limiting step during the cardiac acto-S-1 ATPase cycle just as we previously proposed for the skeletal muscle system. We have also studied the binding of AMP-PNP, PPi, and ADP to skinned muscle fibers and to myofibrils. In both cases, just as we previously observed with acto-S-1, the AMP-PNP and PPi bind weakly but ADP binds strongly. These data support the assumption of our model that the organization of actin and myosin into filament arrays does not affect the binding of nucleotide to the cross-bridge actin complex.